ABSTRACT Up to 50% of urethritis cases, the most common male reproductive tract syndrome, have no known etiology. Imbalances in the microbiota (dysbiosis) are increasingly recognized as factors in the development of clinical syndromes and may cause urethritis. In heterosexual men, Leptotrichia/Sneathia spp., were significantly associated with idiopathic non-gonococcal urethritis (NGU) and men with NGU had distinctive urethral microbial communities (e.g., lower species diversity and higher abundance of Lactobacillis iners and Gardnerella vaginalis than men without NGU), suggesting that the microbiota may play a causal role in the development of urethritis. However, the bacteria that characterized NGU in heterosexual men have been associated with female reproductive tract infection and are likely shared between sex partners. Men who have sex with men (MSM), who comprise half of NGU cases in many settings, rarely, if ever, have exposures to the vaginal microbiota. We hypothesize that the male urethral microbiota reflects the anatomic sites of exposure and that the nature of dysbiosis associated with NGU in MSM differs from heterosexual men. To test this, we will use broad range 16S rRNA gene PCR with high-throughput sequencing to (a) determine how the urethral microbiota that characterizes NGU in MSM differs from that in heterosexual men; (b) determine whether specific microbial communities are associated with persistence and recurrence of NGU in MSM after standard antibiotic therapy; and (c) explore the influence of specific sexual behaviors on the male urethral microbiota and recurrence of NGU. These studies will enhance the clinical management of NGU in the group at highest risk of sexually transmitted infection and HIV, and may lead to novel prevention strategies and/or clinical trials of new therapies to eradicate key bacterial species or restore normal bacteria.